Testicular Leydig cells are the major source of adrogens. These cells contain membrane LH receptors and respond to LH or hCG treatment with increases in cAMP and androgen biosynthesis. In vivo studies have demonstrated the important role of Leydig cells in the maintenance of spermatogenesis and male reproductive organs as well as the ability of various peptide and steroid hormones to regulate Leydig cell functions. Studies on the mechanisms of action to various trophic agents were, however, hampered by the lack of primary cultures of functional Leydig cells for defining the direct effect of these agents on long term Leydig cell functions. We have recently established, for the first time, a functional primary culture of adult testis cells, the androgen production of which was stimulated by hCG for up to 20 days. We propose here to study the role of LH/hCG and cAMP in the regulation of LH receptor and androgen biosyntehsis in primary culture of testis cells. We attempt to purify the Leydig cells and maintain their functions in vitro. The phenomena of "desensitization" by LH and hCG will be studied. We will study the effect of various serum factors and hypophysectomy on Leydig cell LH receptor and steroidogenesis in vitro. We will also study the mechanism of action of several peptide (prolactin, insulin and FSH) and steroid (estrogen, glucocorticoid, androgen and progestin) hormones on Leydig cell functions in vitro. Changes in hormonal responsiveness will be correlated with changes in receptor content for these hormones. The proposed work on the direct hormonal regulation of Leydig cell functions will provide new understanding on the control of testis androgen production and an in vitro model for future development of male pro- and anti-fertility agents.